Journal: Oncogene

Article Title: Helicobacter pylori CagA interacts with E-cadherin and deregulates the beta-catenin signal that promotes intestinal transdifferentiation in gastric epithelial cells.

doi: 10.1038/sj.onc.1210251

Figure Lengend Snippet: Figure 5 Induction of CDX1 and MUC2 in gastric epithelial cells by CagA. (a) WT-A10 cells were cultured in the presence or absence of Dox for 14 days. Total RNAs (20 mg each) prepared were subjected to Northern blotting with the human cdx1 cDNA as a probe. (b) WT-A10 cells or PR-C10 cells were cultured in the presence or absence of Dox for 14 days. As a control, WT-A10 cells were serum- starved for 14 days. Cells were stained with anti-MUC2 antibody (green) and DAPI (blue). (c) WT-A10 cells, p21-inducible cells, b-catenin (S33Y)-inducible cells or p21-b-catenin (S33Y)-double-inducible cells were cultured in the presence or absence of Dox for 14 days. Cells were stained with anti-p21 antibody (red) or anti-b-catenin antibody (red) together with anti-MUC2 antibody (green) and DAPI (blue).

Article Snippet: Antibodies for HA (Y-11), SHP-2 (C-18), cyclin D1 (H-295), anti-p21WAF1/Cip1 (C-19), E-cadherin (G-10), mucin 2 (MUC2) (Ccp58) and b-catenin (H-102) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Cell Culture, Northern Blot, Control, Staining

Journal: Scientific reports

Article Title: Robust and reproducible human intestinal organoid-derived monolayer model for analyzing drug absorption.

doi: 10.1038/s41598-025-95823-z

Figure Lengend Snippet: Fig. 6. Characterization of the novel human duodenal organoid-derived monolayer model. (a) Immunofluorescence analysis for E-cadherin (red), ZO-1 (green), (b) MUC2 (red), and nucleus (blue). Scale bars, 100 µm. (c) Immunofluorescence analysis depicting cross sections of the monolayer model; ZO-1 (green), nucleus (blue), and F-actin (white). Scale bars, 100 µm. Black arrows indicate the Transwell membrane. (d) and (e) Transmission electron microscopic analysis of the monolayer model. Mv, microvilli; Tj, tight junction; Sg, secretory granule-like structures. Scale bars, 10 µm for low magnification images and 2 µm for high magnification images. (f) Efflux transporter P-gp assay using the P-gp substrate digoxin with or without zosuquidar as an inhibitor. AtoB, apical to basal; BtoA, basal to apical. (g) Efflux transporter assay using BCRP substrate sulfasalazine with or without Ko143. (h) UDP-glucuronosyltransferase (UGT) functional assay using raloxifene as a UGT substrate. (i) Carboxylesterase 2 (CES2) functional assay using irinotecan as a substrate with or without loperamide as an inhibitor. The amount of SN-38, a metabolite of irinotecan, was detected in the cells of the monolayer model. (j) and (k) Midazolam permeability and metabolism assay using 2 µM midazolam as a substrate of CYP3A with or without 1-aminobenzotriazole (ABT) as a pan-CYP inhibitor. BLQ, below limit of quantification. (l) Relative gene expression determined by quantitative RT-PCR of the monolayer model with induction of VD3 or VD3 + rifampicin. Cells were treated with inducers for the last two days of the differentiation process. The P value was determined by a two-tailed unpaired t-test.

Article Snippet: The monolayer was incubated with primary antibodies [anti-ZO-1 antibody Alexa Fluor 488 (MA3-39,100-A488; Thermo Fisher Scientific), anti-E-cadherin antibody (PA5-32,178; Thermo Fisher Scientific), and anti-MUC2 antibody (NBP225,221; Novus Biologicals)] in blocking solution containing 0.3% Triton X-100 at 4 °C overnight.

Techniques: Derivative Assay, Immunofluorescence, Membrane, Transmission Assay, Functional Assay, Permeability, Gene Expression, Quantitative RT-PCR, Two Tailed Test

Journal: Saudi Journal of Gastroenterology : Official Journal of the Saudi Gastroenterology Association

Article Title: Aberrant DNA methylation and expression of EYA4 in gastric cardia intestinal metaplasia

doi: 10.4103/sjg.sjg_228_22

Figure Lengend Snippet: Overview of gastric cardiac biopsy specimens and detection rate of gastric cardiac IM. (a) schematic diagram showing the collection of gastric cardiac tissues from cancer-free individuals. A total of 718 participants underwent upper endoscopy were recruited from three endoscopy centers (Shantou, Huizhou, and Shenzhen) in Guangdong Province (b) hematoxylin and eosin staining of representative gastric cardiac tissues from participants with different pathological changes. Scale bars, 50 μm (c) representative images of Alcian blue-periodic acid-Schiff (AB-PAS) staining and immunostaining of mucin-2 (MUC2) in gastric cardiac IM tissues. The AB staining method stains goblet cell mucus as blue color (as indicated by a closed red arrowhead show AB-positive cells); the open red arrowhead denotes epithelial cells expressing MUC2. Scale bars, 50 μm (d) pie chart depicting the detection rates of 718 individuals in our cohort depending on their pathological changes. Left panel: Histologically, gastric cardiac IM was found in 14.1% (101/718) cancer-free individuals. Right panel: A total of four subjects show low-grade intraepithelial neoplasia of the gastric cardiac mucosa in the context of IM. There is mild irregularity of the glands with crowding of epithelial cells. Most of the glandular cells show mucus depletion, the nuclei are elongated, hyperchromatic, and irregular in size. Scale bar, 50 μm (e) boxplot showing the median age in different histological categories. Statistical significance was analyzed by the Kruskal–Wallis test, followed by Dunnett's post hoc test for multiple comparisons between groups. The black horizontal dashed line indicates the median age of all the study participants. The upper and lower limits of the box represent 25th and 75th quartile age distribution with whiskers extending to 1.5 times the range from top/bottom of the box, the center line within the box corresponds to the median age in each group, outliers are not shown (f) line graph showing detection rates of gastric cardiac IM measured in different age and sex groups. Indicated P values were calculated by Pearson's chi-square test. Error bar represents the 95% confidence interval around the detection rate. The overall detection rate of gastric cardiac IM (14.1%) in study participants is depicted by the horizontal dashed black line. Source data (d and f) are provided in Supplementary Table S2

Article Snippet: Slides were incubated with EYA4 primary antibody (Cat# ab251675, rabbit polyclonal, Lot: GR3304276-1, Abcam, UK; 1:100 dilution) or MUC2 primary antibody (Cat# ZM-0392, Lot: 21082308, clone Ccp58, ZSGB-Bio, China; ready-to-use) overnight at 4°C.

Techniques: Staining, Immunostaining, Expressing

Journal: Immunology

Article Title: Human Gut Commensal Bacteroides fragilis Suppresses Mucin Production and Alters Microbiota Composition Resulting in Accelerated Type 1 Diabetes in Mice

doi: 10.1111/imm.70032

Figure Lengend Snippet: Suppressed mucin production in the distal colon of conventionalized BF‐colonised mice. Sections of formalin fixed distal colon and ileum tissues (with luminal contents) of 12‐week‐old conventionalized GF (GF‐conv/Ex‐GF) and conventionalized BF‐monocolonised (BF‐conv/Ex‐BF) mice were subjected to immunofluorescence staining using anti‐Muc2 antibody, or Alcian blue/nuclear fast, periodic acid‐Schiff's/haematoxylin, and Alcian blue periodic acid‐Schiff's/haematoxylin staining. (A) Representative images of immunofluorescence staining of colon and ileum tissue sections (left panels) and relative fluorescence values of Muc2 staining of multiple sections of distal colon tissues from 3 mice/group (right panel) are shown. (B) Representative images Alcian blue/nuclear fast, periodic acid‐Schiff's/haematoxylin, and Alcian blue periodic acid‐Schiff's/haematoxylin staining of colon and ileum tissue sections are shown.

Article Snippet: Tissue sections were stained using a mouse Muc2 specific antibody (Novus Biologicals) followed by an Alexaflor‐488 linked secondary antibody.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: Relative localization and orientation of the 5 gRNAs design for Knock Out of MUC2 on Exon 2 and the primers used for PCR and sanger sequencing.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Knock-Out, Sequencing

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: Volcano plot of the DEG using limma between mucinous versus all other histological classifications in hypermutated samples of the AC-ICAM cohort. The most significant gene expressed in mucinous samples with the higher fold change is MUC2 (p = 5.38e-16, logFC = 3.50). Two other mucins are identified as upregulated in mucinous samples: MUC5B (p = 5.40e-11, logFC = 2.50) and MUC6 (p = 2.80e-9, logFC = 2.63). Red dotes represents genes with p-value < 0.005 and grey dotes for genes with p-value > 0.005.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques:

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. Westernblot of full length MUC2 protein (540kDa) and beta actin (housekeeping gene, 43kDa) in 3 different cell lines LS-174T, HT-29 and LoVo grown in 2D (N=3 biological replicates). B. Quantification of band intensity of MUC2 protein in those 3 cells lines grown in 2D. MUC2 is present in abundance in LS-174T, with significantly less expression in HT-29 (p=0.0019) (unpaired T-test) and no expression in LoVo cell line, is observed. C. Relative mRNA expression of MUC2 analyzed by Q-RT-PCR (N=3 biological replicates) in the 3 cell line grown in 2D. MUC2 expression is strongest in LS-174T compared to HT-29 (p=0.0002) (unpaired T-test) and no expression in LoVo is observed. D. Westernblot of full length MUC2 protein (540kDa) and beta actin (43 kDa) in 3 different cell lines LS-174T, HT-29 and LoVo grown in 3D (N=3 biological replicates). E. Quantification of band intensity of MUC2 protein in those 3 cells lines grown in 2D. MUC2 is present in abundance in LS-174T, with significantly less expression in HT-29 (p<0.0001) (unpaired T-test) and no expression in LoVo cell line, is observed. F. Relative mRNA expression of MUC2 analyzed by Q-RT-PCR (N=3 biological replicates) in the 3 cell line grown in 2D. MUC2 expression is strongest in LS-174T compared to HT-29 (p=0.0002) (unpaired T-test) and no expression in LoVo is observed.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. Westernblot of full length MUC2 protein (540kDa) and beta actin (43 kDa) in 3 different cell lines LS-174T CTRL, LS-174T MUC2 K.O., HT-29 CTRL and HT-29 MUC2 K.O. grown in 2D (N=3 biological replicates). B. Westernblot of full length MUC2 protein (540kDa) and beta actin (43 kDa) in 3 different cell lines LS-174T CTRL, LS-174T MUC2 K.O., HT-29 CTRL and HT-29 MUC2 K.O. grown in 2D (N=3 biological replicates). C. Representative images of spheroids of HT-29 and LS-174T CTRL and MUC2 K.O. immunostaining with Mouse anti-hMUC2 antibody (green) and counterstained with DAPI. Images acquired on confocal LS780. Scale bar of 100µm.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Immunostaining

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. HT-29 CTRL and HT-29 MUC2 K.O. growth in 2D culture setup monitored by plate reader for 7 days by GFP intensity (N=3 biological replicates in 30 technical replicate each). HT-29 CTRL grow significantly faster than HT-29 MUC2 K.O. (p=0.0011) (unpaired t-test). B. LS-174T CTRL and LS-174T MUC2 K.O. growth in 2D culture setup monitored by plate reader for 7 days by GFP intensity (N=3 biological replicates in 30 technical replicate each). No significant difference was observed (unpaired t-test). C. HT-29 CTRL and HT-29 MUC2 K.O. growth in 2D culture setup monitored by plate reader for 7 days by GFP intensity (N=3 biological replicates in 30 technical replicate each). No significant difference was observed (unpaired t-test). D. LS-174T CTRL and LS-174T MUC2 K.O. growth in 2D culture setup monitored by plate reader for 7 days by GFP intensity (N=3 biological replicates in 30 technical replicates each). LS-174T CTRL grew significantly faster than LS-174T MUC2 K.O. (p=0.0134) (unpaired t-test). E. Representative images of day 5 spheroids acquired with zeiss CD7 fluorescent microscope of GFP and oblique of spheroids of HT-29 and LS-174T CTRL or MUC2 K.O. Scale of 200µm. F. Roundness analysis of HT-29 (N=13 each, biological replicates) and LS-174T (N=25 each, biological replicates) of CTRL and MUC2 K.O. performed by analysis with Zen blue software of area and perimeter of each spheroid. No significant difference is observed between HT-29 CTRL and HT-29 MUC2 K.O., but LS-174T MUC2 K.O. is significantly more round than LS-174T CTRL (unpaired t-test).

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Microscopy, Software

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. Surface area analysis of HT-29 CTRL and HT-29 MUC2 K.O. spheroids on day 5 (N=12 biological replicates). B. Surface area analysis of LS-174T CTRL and HT-29 MUC2 K.O. spheroids on day 5 (N=12 biological replicates).

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques:

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. Box plot of adherence junction enrichment score (ES) in HT-29 and LS-174T. The ES is up-regulated in HT-29 MUC2 K.O. compared to CTRL (p=0.041) (student t-test). In contrast, the ES is down-regulated in LS-174T MUC2 K.O. compared to CTRL (p=0.047) (student t-test). B. Representative capillary western blot (CWB) results of E-cadherin, N-cadherin and Vimentin expression in human fetal fibroblast (HFF), Hela, HT-29 CTRL and HT-29 MUC2 K.O. (N=2 technical replicates). C. Representative capillary western blot (CWB) results of E-cadherin, N-cadherin and Vimentin expression in HFF, Hela, LS-174T CTRL and LS-174T MUC2 K.O. (N=2 technical replicates). D. Quantification of band intensity of E-cadherin, N-cadherin and Vimentin normalized to beta actin expression in HFF, Hela, HT-29 CTRL and HT-29 MUC2. (N=2, technical replicates). E. Quantification of band intensity of E-cadherin, N-cadherin and Vimentin normalized to beta actin expression in HFF, Hela, LS-174T CTRL and LS-174T MUC2 K.O.. (N=2, technical replicates).

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. Representative micrographs of one of the individual experiments at time points 0, 24, 48 and 72 h in HT-29 CTRL and HT-29 MUC2 K.O. No cell motility toward the gap area was seen even after 72h, despite a clear cell multiplication. B. Size of scratch in HT-29 CTRL and MUC2 K.O. relative to 0 hour (N=12). C. Representative micrographs of one of the individual experiments at time points 0, 24, 48 and 72 h in LS-174T CTRL and LS-174T MUC2 K.O. No cell motility toward the gap area was seen even after 72h, despite a clear cell multiplication. D. Size of scratch in LS-174T CTRL and MUC2 K.O. relative to 0 hour (N=16).

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques:

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: Comparison of epithelial mesenchymal transition enrichment score between CTRL (2D) and MUC2 K.O. (2D), and between CTRL (3D) and MUC2 K.O. (3D) in A. HT-29, and B. LS-174T. P-value from student t-test.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Comparison

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. HT-29 CTRL and MUC2 K.O. growth and death monitored by GFP expression over 7 days in 2D set up. Top panel shows the GFP reading for HT-29 CTRL only with 0 to 10.000 E/A PBMCs added on day 2 the following panel represents the comparison of HT-29 CTRL and HT-29 MUC2 K.O. with a defined number of E/A PBCMs. Data are presented as relative to the same cell line without co-culture with PBMCs (N=18, 3 biological replicates of 6 technical replicates). B. LS-174T CTRL and MUC2 K.O. growth and death monitored by GFP expression over 7 days in 2D set up. Top panel shows the GFP reading for LS-174T CTRL only with 0 to 10.000 E/A PBMCs added on day 2 the following panel represents the comparison of LS-174T CTRL and LS-174T MUC2 K.O. with a defined number of E/A PBCMs. Data are presented as relative to the same cell line without co-culture with PBMCs (N=18, 3 biological replicates of 6 technical replicates). C. HT-29 CTRL and MUC2 K.O. growth and death monitored by GFP expression over 9 days in 3D set up. Top panel shows the GFP reading for HT-29 CTRL only with 0 to 10.000 E/A PBMCs added on day 5 the following panel represents the comparison of HT-29 CTRL and HT-29 MUC2 K.O. with a defined number of E/A PBCMs. Data are presented as relative to the same cell line without co-culture with PBMCs (N=18, 3 biological replicates of 6 technical replicates). D. LS-174T CTRL and MUC2 K.O. growth and death monitored by GFP expression over 7 days in 3D set up. Top panel shows the GFP reading for LS-174T CTRL only with 0 to 10.000 E/A PBMCs added on day 5 the following panel represents the comparison of LS-174T CTRL and LS-174T MUC2 K.O. with a defined number of E/A PBCMs. Data are presented as relative to the same cell line without co-culture with PBMCs (N=18, 3 biological replicates of 6 technical replicates). E. Comparison of HT-29 with LS-174T CTRL or MUC2 K.O. with various number of E/A PBMCs in 2D. Data are presented as relative to the same cell line without co-culture with PBMCs (0 E/A PBMCs) (N=18, 3 biological replicates of 6 technical replicates). F. Comparison of HT-29 with LS-174T CTRL or MUC2 K.O. with various number of E/A PBMCs in 3D. Data are presented as relative to the same cell line without co-culture with PBMCs (0 E/A PBMCs) (N=18, 3 biological replicates of 6 technical replicates).

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Expressing, Comparison, Co-Culture Assay

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. HT-29 and LS-174T, CTRL and MUC2 K.O. growth and death monitored by GFP expression over 7 days in 2D and 9 days in 3D culture set up. 0 or 500 E/A PBMCs were added on day 2 for 2D and 0 or 1000 E/A PBMCs were added on day 5 for 3D co-culture setup. Data are presented as relative to the same cell line without co-culture with PBMCs (0 E/A PBMCs) (N=18, 3 biological replicates of 6 technical replicates). Significant differences between HT-29 and LS-174T cell lines were calculated at day 7 in 2D and 3D by student T-test. B. HT-29 CTRL and MUC2 K.O. spheroids area of IN fraction spheroids post rinse after co-culture for 2 days with E/A PBCMs relative to area of IN fraction after rinse without co-culture. After co-culture with 2000 or 4000 PBMCs area of IN fraction spheroids is significantly smaller in HT-29 MUC2 K.O. than in HT-29 CTRL (N=12, 2 biological of 6 technical replicates) (unpaired t-test). C. Representative images of IN fraction spheroids post rinse after co-culture with 4000 E/A PBMCs of HT-29 CTRL and HT-29 MUC2 K.O. Images were acquired for oblique and GFP expression on Zeiss CD7, and area analyzed with Imaris software. Scale bar of 200µm. D. LS-174T CTRL and MUC2 K.O. spheroids area of IN fraction spheroids post rinse after co-culture for 2 days with E/A PBCMs relative to area of IN fraction after rinse without co-culture. No significant difference was observed between LS-174T CTRL and MUC2 K.O. at any number of E/A PBMCs co-culture (N=12, 2 biological of 6 technical replicates) (unpaired t-test). E. Representative images of IN fraction spheroids post rinse after co-culture with 4000 E/A PBMCs of LS-174T CTRL and LS-174T MUC2 K.O. Images were acquired for oblique and GFP expression on Zeiss CD7, and area analyzed with Imaris software. Scale bar of 200µm.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Expressing, Co-Culture Assay, Software

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: Representative video of HT-29 CTRL, HT-29 MUC2 K.O., LS-174T CTRL, LS-174T MUC2 K.O. from day 5 to day 7 with and without E/A PBMCs.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques:

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. Average number of Epcam+ cells recovered in the IN fraction after digestion, staining and flow analysis without co-culture with E/A PBMCs. Significantly less Epcam+ cells were recovered for HT-29 CTRL than for HT-29 MUC2 K.O. (p=0.0182, Paired T-test) (N=4 biological replicates). B. Average number of CD45+ cells recovered in the IN fraction after digestion, staining and flow analysis. Significantly less CD45+ cells were recovered for HT-29 CTRL than for HT-29 MUC2 K.O. (p=0.0433, Paired T-test) (N=4 biological replicates). C. Average number of CD45+ cells recovered in the OUT fraction after digestion, staining and flow analysis. No significant difference in CD45+ cells recovered after co-culture with HT-29 CTRL or HT-29 MUC2 K.O. was detected despite having systematically more CD45+ cells after co-culture with HT-29 MUC2 K.O. than with HT-29 CTRL (p=0.1460, Paired T-test) (N=4 biological replicates). D. Pie chart representation of average relative abundance of Epcam+ and CD45+ cells in the IN and OUT fraction after 2 days of co-culture for HT-29 CTRL and MUC2 K.O. (N=4 biological replicates). E. Average number of Epcam+ cells recovered in the IN fraction after digestion, staining and flow analysis without co-culture with E/A PBMCs. No statistical difference was observed in Epcam+ cells recovered between LS-174T CTRL and LS-147T MUC2 K.O. (p=0.1405, Paired T-test) (N=3 biological replicates). F. Average number of CD45+ cells recovered in the IN fraction after digestion, staining and flow analysis. No difference in CD45+ cells recovered was observed between LS- 174T CTRL and LS-174T MUC2 K.O. (p=0.9202, Paired T-test) (N=4 biological replicates). G. Average number of CD45+ cells recovered in the OUT fraction after digestion, staining and flow analysis. No significant difference in CD45+ cells recovered after co-culture with LS-174T CTRL or LS-174T MUC2 K.O. was detected despite having systematically more CD45+ cells after co-culture with LS-174T CTRL. than with LS-174T MUC2 K.O. (p=0.1262, Paired T-test) (N=4 biological replicates). H. Pie chart representation of average relative abundance of Epcam+ and CD45+ cells in the IN and OUT fraction after 2 days of co-culture for LS174T CTRL and MUC2 K.O. (N=4 biological replicates).

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Staining, Co-Culture Assay

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: A. Principal component analysis (PCA) based on gene expression profile in HT-29, LS- 174T cultured in 2D and 3D, E/A PBMCs or IN and OUT fraction of co-culture of cancer cells with E/A PBMCs. B: Venn diagram of DEG (FDR < 0.01, logFC >= 1) between HT-29 MUC2 K.O. + E/A PBMCs IN Fraction vs. HT-29 CTRL + E/A PBMCs IN fraction (n = 2073 genes) and HT- 29 MUC2 K.O. (3D) vs. HT-29 CTRL (3D) (n = 345). Pathways enrichment analysis was performed on genes reflecting DEG between PBMCs IN (MUC2) vs. PBMCs IN (CTRL) (n = 1950) by subtracting the common genes. Top 20 pathways based on p-value were selected for plotting. Comparison of those two DEG lists allows us to see that when in contact with HT-29 MUC2 K.O., E/A PBMCs increase their cell cycle and are IFN pathway activation. C. Venn diagram of DEG (FDR < 0.01, logFC >= 1) between LS-174T MUC2 + E/A PBMCs IN fraction vs. LS-174T CTRL + E/A PBMCs IN fraction (n = 891) and MUC2 K.O. (3D) vs. CNTL (3D) (n = 463). Pathways enrichment analysis was performed on genes reflecting DEG between PBMCs IN (MUC2) vs. PBMCs IN (CTRL) (n = 655) by subtracting common the common genes. Top 20 pathways based on p-value were selected for plotting (FDR < 0.01). Comparison of those two DEG lists does not demonstrate similar pattern with LS-174T. D. Genes expression of other gel forming mucin in HT-29 and LS-174T spheroids shows that LS-174T also have strong expression of MUC5B, MUC6 and MUC19 compared to HT-29. A and B. Histograms represent the proportion (%) of DEGs upregulated (red) or downregulated (green) in PBMCs IN (MUC2) vs. PBMCs IN (CTRL). The circles represent the pathway activation status. Blue circle indicates the pathway is inhibited with a negative z-score, orange circle represents a pathway is activated with a positive z-score, the white circle represents the pathway is neutral with zero z-score, while a gray circle indicates that the pathway activity is unknown.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Expressing, Cell Culture, Co-Culture Assay, Comparison, Activation Assay, Activity Assay

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: IPA pathways enrichment analysis. Top 20 significant pathways based on p-value associated with DEG (FDR < 0.01, logFC >=1) between MUC2 K.O. (2D) and CTRL (2D) in A. HT-29 (n = 557) C. LS-174T (n = 1636). Between MUC2 K.O (3D) and CTRL (3D) in B. HT-29 (n = 345) D. LS-174T (n = 463). Histograms represent the proportion (%) of DEGs upregulated (red) or downregulated (green) in MUC2 K.O versus CTRL. The circles represent the pathway activation status. Blue circle indicates the pathway is inhibited with a negative z-score, orange circle represents a pathway is activated with a positive z-score, the white circle represents the pathway is neutral with zero z-score, while a gray circle indicates that the pathway activity is unknown.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Activation Assay, Activity Assay

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: Comparison of ICR score mean value between CTRL + E/A PBMCs IN fraction and MUC2 K.O. + E/A PBMCs IN fraction in HT-29 and LS-174T. P-value from student t-test.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Comparison

Journal: bioRxiv

Article Title: MUC2 Expression Modulates Immune Infiltration in Colorectal Cancer

doi: 10.1101/2024.08.06.594842

Figure Lengend Snippet: Comparison of genes expression of other gel forming mucin in HT-29 and LS-174T spheroids between MUC2 K.O. (in blue) and CTRL (in black) (3D). Statistical analysis represented as p-values from student’s t-test.

Article Snippet: MUC2 antibody (CCP58) (Novus #NBP2-25221) and HRP-conjugated β actin monoclonal antibody (Proteintech, #HRP-60008) were used as primary antibody as well as Anti-mouse IgG (H+L) antibody, human serum adsorbed and peroxidase-labeled (Seracare, # 5450-0011) as secondary anti body.

Techniques: Comparison, Expressing